Mutant Huntingtin Impairs Astrocytic Secretion Open Access

Abstract

Huntington's disease (HD) is a fatal, inherited, neurodegenerative disorder that affects one in every 10,000 Americans. To date, there is no effective treatment, in part because the pathogenic mechanism driving the disease is not fully understood. Expansion of a polyglutamine (polyQ) repeat in the N-terminal region of the HD protein, huntingtin (Htt), is the molecular basis of the disease. Although neuronal cells are preferentially degenerated in HD, the function of glial cells is also affected by mutant huntingtin (mHtt). Astrocytes and other glial cells can release a variety of functional cellular vesicles into the extracellular space to support the normal function and survival of neighboring neurons. My thesis work focused on two types of important vesicles, brain-derived neurotrophic factor (BDNF)-containing dense-core vesicles and heat shock protein-containing exosomes. Exosomes are small membranous vesicles that are secreted by multiple cell types and carry protein and genetic materials for cell-cell communication. Whether mHtt affects astrocytic secretion in the HD brain remains unknown. We used full-length HD knock-in (KI) mice to investigate the effect of mHtt on astrocytic secretion. Our results found that mHtt binds Rab3a, a small GTPase localized on membranes of dense-core vesicles, to affect its GTP/ GDP exchange; this impairs the docking of dense-core vesicles in HD astrocytes, resulting in decreased release of BDNF. Importantly, overexpression of Rab3a rescues impaired BDNF vesicle docking and secretion from astrocytes and alleviates HD early neuropathology in the HD KI mouse brain. Our results also demonstrated that mHtt reduces the expression of αB-crystallin, a small heat shock protein that mediates exosome secretion, in astrocytes. This effect reduces exosome secretion in HD brains, which could be alleviated by overexpression of αB-crystallin. These findings have revealed new pathological pathways that affect astrocytic secretion and contribute to non-cell-autonomous neurotoxicity in HD.

Table of Contents

Table of Contents

Chapter 1: General Introduction………………………………………................ 1

1.1 Polyglutamine Disease.……….…………………………………………… 2

1.2 Huntington's Disease……………………………………………………… 3

1.3 Glial Dysfunction in Huntington's Disease……………………………...... 6

1.4 Neurotrophic Factors in Huntington's Disease……………………………. 9

1.5 Exocytosis in Astrocytes………………………………………………….. 11

1.6 Rab proteins in Huntington's Disease…………………………………….. 13

1.7 Roles of Exosomes in Neuron-Glia Communication……………………….15

1.8 Dissertation Goals…………………………………………………………..17

Chapter 2: Materials and Methods……………………………………….. 27

2.1 Animals…………………………………………………………………….. 28

2.2 Antibodies and Reagents…………………………………………………… 28

2.3 Primary Cultures…………………………………………………………..... 29

2.4 Preparation of ACM………………………………………………………… 29

2.5 Enzyme-Linked Immunosorbent Assays (ELISA)…………………………. 30

2.6 Western Blotting……………………………………………………………. 30

2.7 Glutamate Measurement…………………………………………………… 30

2.8 RT-qPCR.…………………………………………………………………... 31

2.9 Knockdown Assay…………………………………………………………. 31

2.10 Stereotaxic Injection of Virus…………………………………………….. 31

2.11 Preparation of Brain Slices………………………………………………... 32

2.12 Rab3 GTPase Activity Assay………………………………………………32

2.13 Total Internal Reflection Fluorescence (TIRF) microscopy……………..... 33

2.14 Immunoprecipitation………………………………………………………. 34

2.15 Immunofluorescence Staining…………………………………………….. 34

2.16 ATP Assay………………………………………………………………… 35

2.17 Purification of Exosomes…………………………………………………. 35

2.18 Electron Microscopy and Immunogold Labeling……………………….... 36

2.19 Chromatin Immunoprecipitation (ChIP)………………………………….. 37

2.20 Statistical Analyses……………………………………………………….. 37

Chapter 3: Mutant Huntingtin Impairs BDNF Release from Astrocytes by Disrupting Conversion of Rab3a-GTP into Rab3a-GDP…………………… 38

3.1 Abstract……………………………………………………………………. 39

3.2 Introduction………………………………………………………………… 40

3.3 Results……………………………………………………………………… 41

3.4 Discussion………………………………………………………………….. 50

Chapter 4: Mutant Huntingtin Inhibits αB-crystallin Expression and Impairs Exosome Secretion From Astrocytes…………………………………………… 73

4.1 Abstract……………………………………………………………………... 74

4.2 Introduction…………………………………………………………………. 74

4.3 Results……………………………………………………………………… 76

4.4 Discussion………………………………………………………………….. 83

Chapter 5: General Conclusions and Future Directions…………………….. 103

5.1 Summary…………………………………………………………………….104

5.2 Future Directions……………………………………………………………109

5.3 Conclusions………………………………………………………………… 112

References……………………………………………………………………………116

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience
Keyword	neurotrophic factors Glia neurodegeneration exocytosis
Committee Chair / Thesis Advisor	Li, Xiao-Jiang, Emory University Li, Shi Hua, Emory University
Committee Members	Walker, Lary C, Emory University Hepler, John R, Emory University Kukar, Thomas, Emory University Tansey, Malu, Emory University

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